Spectrophotometric method and apparatus for the non-invasive

ABSTRACT

A directive light beam in a wavelength range appropriate for penetrating into body tissues is applied to a portion of a patient&#39;s body and the energy transmitted or back-scattered by the underlying tissue is analyzed spectrophotometrically for the presence of glucose. Analysis is performed using especially selected bands in the near-infrared region.

FIELD OF INVENTION

The present invention concerns the photometric determination of glucosein the bloodstream or tissues of patients suspected to have developeddiabetes. This determination is carried out by measuring the opticalnear infrared absorption of glucose in regions of the spectrum wheretypical glucose absorption bands exist and computing the measured valueswith reference values obtained from regions of the spectrum whereglucose has no or little absorption and where the errors due tobackground absorptions by the constituents of the surrounding tissues orblood containing the glucose are of reduced significance or can bequantitatively compensated.

BACKGROUND OF THE ART

Many methods and devices have been developed up to now for thedetermination of glucose in vitro or in vivo by optical means.

For instance, in PCT application WO No. 81/00622, there is disclosed anIR absorption method and apparatus for determining glucose in bodyfluids. According to this reference, absorption spectra of serum orurine, both transmissive or reflective, i.e. due to back-scatteringeffects, are measured at two distinct wavelengths λ1 and λ2, λ2 beingtypical of the absorption of a substance to be measured (for instanceglucose) and λ1 being a wavelength at which the absorption is roughlyindependent of the concentration of the substance of interest. Then thepertinent measured data are derived from calculating the ratio of theabsorption values at λ1 and λ2, the bands of interest being in the rangeof 940-950 cm⁻¹ (10.64-10.54 μm) and 1090-1095 cm⁻¹ (9.17-9.13 μm),respectively. In this reference, the source of irradiation is providedby a CO₂ laser.

Swiss Pat. No. CH-612.271 discloses a non invasive method to determinebiological substances in samples or through the skin using an attenuatedtotal reflection (ATR) prism. This method relies on the passing of aninfrared beam through a wave-guide (ATR prism) directly placed against asample to be analyzed (for instance the lips or the tongue). Therefractive index of the wave-guide being larger than that of the mediumof the sample (optically thinner medium), the beam propagates thereinfollowing a totally reflected path, the only interaction thereof withsaid thinner medium (to be analyzed) being that of the "evanescent wave"component at the reflection site (see also Hormone & MetabolicRes./suppl. Ser. (1979), p. 30-35). When using predetermined infraredwavelengths typical of glucose absorption, the beam in the ATR prism isattenuated according to the glucose concentration in the opticallythinner medium, this attenuation being ascertained and processed intoglucose determination data. U.S. Pat. No. 3,958,560 discloses anon-invasive device for determining glucose in patient's eyes. Suchdevice comprises a contact-lens shaped sensor device including aninfrared source applied on one side of the cornea and a detector on theother side thereof. Thus, when a infrared radiation is applied to thearea under measurement, light is transmitted through the cornea and theaqueous humor to the detector. The detected signal is transmitted to aremote receiver and a read-out device providing data on theconcentration of glucose in the patient's eye as a function of thespecific modifications undergone by the IR radiations when passingthrough the eye.

GB patent application No. 2,033,575 discloses a detector device forinvestigating substances in a patient's regions near to the bloodstream,namely CO₂, oxygen or glucose. The key features of such detectorcomprise radiation directing means arranged to direct optical radiationinto the patient's body, and receiver means for detecting attenuatedoptical radiations backscattered or reflected within the patient's bodyi.e. from a region below the skin surface. The detected signal isthereafter processed into useful analytical data. Optical radiationsinclude UV as well as IR radiations.

Other references rather refer to the measurement or monitoring of otherbioactive parameters and components such as blood flow, metabolicoxyhemoglobin and desoxyhemoglobin but, in reason of their closeanalogies with the aforementioned techniques, they are also worthreviewing here. Thus U.S. Pat. No. 3,638,640 discloses a method and anapparatus for measuring oxygen and other substances in blood and livingtissues. The apparatus comprises radiation sources and detectorsdisposed on a patient's body, for instance about the ear to measure theintensity passing therethrough or on the forehead to measure theradiation reflected therefrom after passing through the blood and skintissue. The radiations used belong to the red and very near infraredregion, for instance wavelengths (λ) of 660, 715 and 805 nm. The numberof different wavelengths used simultaneously in the method is equal tothe total of at least one measuring wavelength typical for eachsubstance present in the area under investigation (including thesubstance(s) to be determined) plus one. By an appropriate electroniccomputation of the signals obtained after detection from absorption atthese diverse wavelengths useful quantitative data on the concentrationsof the substance to be measured are obtained irrespective of possiblechanges in measurement conditions such as displacement of the testappliance, changes in illumination intensity and geometry, changes inthe amount of blood perfusing the tissue under investigation and thelike.

GB patent application No. 2,075,668 describes a spectrophotometricapparatus for measuring and monitoring in-vivo and non-invasively themetabolism of body organs, e.g. changes in the oxido-reduction state ofhemoglobin and cellular cytochrome as well as blood flow rates invarious organs such as brain, heart, kidney and the like. The aboveobjects are accomplished by optical techniques involving wavelengths inthe 700-1300 nm range which have been shown to effectively penetrate thebody tissues down to distances of several mm. Thus in FIG. 14 of thisreference there is disclosed a device involving reflectance typemeasurements and comprising a light source for injecting light energyinto a waveguide (optical fiber bundle) applied to the body and disposedin such way (slantwise relative to the skin) that the directionallyemitted energy whch penetrates into the body through the skin isreflected or back scattered by the underlying tissue to be analyzed atsome distance from the source; the partially absorbed energy thenreaches a first detector placed also over the skin and somewhatdistantly from the source. Another detector placed coaxially with thesource picks up a back radiated reference signal, both the analyticaland reference signals from the detectors being fed to a computingcircuit, the output of which provides useful read-out data concerningthe sought after analytical information.

Although the aforementioned techniques have a lot of merit somedifficulties inherent thereto still exist. These difficulties are mainlyrelated to the optical properties of the radiations used for making themeasurements. Thus, radiation penetration into the skin depends on theaction of absorbing chromophores and is wavelength-dependent, i.e. thelight in the infrared range above 2.5 μm is strongly absorbed by waterand has very little penetration capability into living tissuescontaining glucose and, despite the highly specific absorption of thelatter in this band, it is not readily usable to analyze body tissuevolumes at depths exceeding a few microns or tens of microns. Ifexceptionally powerful sources (i.e. CO₂ laser) are used, deeperpenetration is obtained but at the risk of burning the tissues underexamination. Conversely, using wavelengths below about 1 micron (1000nm) has the drawback that, although penetration in this region is fairlygood, strong absorbing chromophores still exist such as hemoglobin,bilirubin ad melanin and specific absorptions due to glucose areextremely weak which provides insufficient or borderline sensitivity andaccuracy for practical use in the medical field. In addition, the ATRmethod which tries to circumvent the adverse consequences of the heateffect by using the total internal reflection technique enables toinvestigate depths of tissues not exceeding about 10 μm which isinsufficient to obtain reliable glucose determination information.

DISCLOSURE OF THE INVENTION

The present invention remedies these shortcomings. Indeed it was foundquite unexpectedly that by operating at some wavelengths located in the1000 to 2500 nm range, acceptable combinations of sufficient penetrationdepth to reach the tissues of interest and sufficient sensitivity inascertaining glucose concentration variations could be accomplished,this being without risks of overheating tissues. At such penetrationdepths of, say, 0.5 mm to several mm, representative information on theconditions of patients could be gained in regard to possible lack orexcess of glucose in the blood stream (hypo- or hyperglycemia).Therefore, one object of the invention is a spectrophotometric methodfor the transcutaneous non-invasive determination of glucose in patientssuffering or suspected to suffer from diabetes in which a portion ofsaid patient's body is irradiated with the light of a directionaloptical lamp source, the resulting energy I either transmitted ordiffusely reflected (back-scattered) by a sample volume of body tissueunderneath the skin of said body portion being collected and convertedinto suitable electrical signals, said collected light including atleast one spectral band of a first kind containing a measuring signalwavelength λG typical of the glucose absorption and at least anotherband of a second kind with a reference signal wavelength λR typical ofthe background absorption spectrum due to the tissue containing theglucose but in which the absorption of the latter is nil orinsignificant, and in which method said electrical signals (the value ofwhich, IG and IR, are representative of the absorption in said measuringand reference bands, respectively) are fed to an electronic computingcircuit for being transformed into glucose concentration readouts,characterized in that the bands of the first and second kind below tothe 1000 to 2500 nm medium near-IR range, λG being selected from 1575,1765, 2100 and 2270+ or -15 nm and λR being selected either in the range1100 to 1300 nm or in narrow regions situated on both sides of themeasuring bands but outside the area where glucose absorbs strongly.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 represents schematically the main components of an apparatus fornon-invasively measuring glucose in vivo by an absorptive transmissiontechnique.

FIG. 2 represents schematically a detail of a variant of the apparatusof FIG. 1 designed to operate by an absorptive reflective technique.

FIG. 3 represents schematically the components for processing theelectrical signals obtained from the light gathered after being partlyabsorbed in the region of interest and for computing and converting saidsignals into useful readouts of glucose determination.

FIG. 4 represents a plot of absorption measurement data versus glucoseconcentration at both λG=2100 nm and λR=1100 nm.

FIG. 5 represents an infrared spectrum of glucose (1 mole/1 aqueoussolution) from which the corresponding infrared spectrum of water hasbeen subtracted.

FIG. 6 is like FIG. 5 but refers to blood serum and water.

FIG. 7 is like FIG. 5 but refers to human serum albumin.

FIG. 8 is like FIG. 5 but refers to keratin.

FIG. 9 is like FIG. 5 but refers to collagen.

FIG. 10 is like FIG. 5 but refers to HCO₃ ⁻.

The light absorbed by the tissue subjected to analysis constitutestogether with other losses due to scattered stray radiations inherent tothe practice of the method and the apparatus components, the backgroundresponse noise from which the useful signals must be separated. Theabsorbing entities in the body media containing the glucose includepeptidic substances such as albumin, keratin, collagen and the like aswell as low molecular weight species such as water, hydrogenocarbonate,salt and the like. These substances all have characteristic absorptionsdistinct from the aforementioned selected typical glucose absorptions asshown by the infrared spectra of FIGS. 5 to 10; and compensation canthus be afforded by subjecting the collected measuring and referencedata to computation according to programmed algorithms. Further, thetime concentration variation of the components depicted in FIGS. 5 to 10in the blood and/or living tissues follows a pattern different from thatof glucose in the measurement location, which difference is also usableto determine glucose in the presence of such components. Examples ofpossible computation algorithms are provided in the following reference:R. D. ROSENTHAL, an Introduction to Near Infrared Quantitative Analysis,1977, Annual Meeting of American Association of Cereal Chemists.

According to one general method of computing applicable in the presentinvention a normalizing factor is first established from the differencesin absorptions in the reference band when glucose is present or absentor in insignificant quantities in the tissue to be analyzed. Then thisfactor is used to normalize the measured value of glucose absorption inthe λG band, the reference value being subtracted from the normalizedvalue to provide the data for expressing the correct glucoseconcentration in the sample. The normalizing factor can be determinedfor instance by setting the reference's wavelength at an isosbesticpoint (i.e. a wavelength at which there is no significant change inabsorption although the concentration of glucose may change).

Another way to obtain a normalizing factor is to focus alternately fromthe place where glucose should be analyzed to a place where the amountof glucose is either insignificant or constant and fairly well known,the background absorption spectrum being substantially constant orcomparably shaped in the two locations. One will see hereinafter howthis can be practically implemented.

According to another way of computing the absorption measured valuesinto useful glucose determination results is to differentiate the IG andIR signals with regard to λ within the area of the bands of the firstand of the second kind, respectively; and then to subtract onedifferential from the other and obtain the desired result from thedifference. Reference to this method is provided in T. C. O'HAVERPotential Clinical Applications of Derivative and Wavelength ModulationSpectrometry, Clin. Chem. 25(a), 1548-53 (1959).

The invention also concerns an apparatus for carrying out the presentanalytical method.

This apparatus comprises a light source for directively applying a beamof light on a portion of a patient's body, the spectral composition ofsaid light being such that it can penetrate through the skin to a regionwhere glucose concentration can be measured with significance regardingthe patient's conditions and from which said light can be gathered afterbeing partially absorbed as a function of the glucose concentration, acollecting means for gathering the radiation transmitted or reflected(transflected) from said region, detector means for detecting andconverting into electrical signals the gathered light as distinctwavelengths belonging to at least two bands, one measuring band and onereference band, and computing means to transform said electrical signalsfrom useful readouts representative of the desired glucose measurementdata. One characteristic feature of an embodiment of this apparatus isthat it comprises means for varying continuously or stepwise theincidence angle relative to the body of said beam of light, saidvariation resulting in a consequent variation of the depth of the centerof said region wherefrom the light is gathered after absorption.

Such an apparatus and variants thereof will now be described with thehelp of the annexed drawing.

The apparatus represented in FIG. 1 consists of two main sections, alight source section 1 and a detector section 2. The light sourcesection 1 comprises a light source 3, for instance a halogen lamp andlight directing means, for instance a reflector 4 and a condensor 5 forproviding a directed beam 6 of light. This beam needs not be polarizedor coherent but, of course, such types of light can also be used ifdesired. When using a wide band continuous spectrum of light, theapparatus also comprises a filter or system of filters 7 to block outundesired wavelength ranges mainly caused by higher order diffraction atthe monochromator grating; in this particular application where thesignals should be in the range of about 1000 to 2700 nm, visible rangesare eliminated by using a SCHOTT RG780 filter (0.8-1.3 μm), a siliconfilter (1.3-2.2 μm) and a Ge filter (2.2-4.2 μm). The lamp is a 12 V,100 W halogen lamp with the following properties: color temperature3300° K.; maximum output at 850 nm; average luminance 3500 cd/cm². Ofcourse, if monochromatic light sources were used in the presentapparatus, for instance by means of tunable lasers, the blocking filters7 would no longer be necessary.

The apparatus further comprises a monochromator 8 with inlet slit 8a andoutput slit 8b and a grating 8c. The monochromator can be of anysuitable origin, for instance a JARREL-ASH model 70000 with sine barwavelength drive is suitable. The monochromator can scan orrepeatitively shift from one selected wavelength to another one or, insuccession, to several ones depending on whether one or more measuringand references wavelengths are used concurrently in the analysis. Theshifting or scanning rate of the monochromator is programmed andcontrolled by the computer circuits to be described later and thesignals thereto are provided by line 12a. Of course, if the source lightis provided by means of lasers of specific wavelengths, themonochromator is no longer necessary.

The selected monochromatic beam 9 which emerges from the monochromatorpasses through a chopper disk 10 driven by a motor 11 whose rotation iscontrolled by a clock (not represented but conventionally inherent toany chopper system); this system also provides timing signals, theoutput of which is schematically represented by the arrow 12b, to beused for synchronizing the analog and digital electronic processingcircuits as will be seen hereinafter. The periodical interruption of theexcitation beam of light 9 by the chopper disk is required for removingor minimizing the background noise due to ambient light, detector darknoise, and other stray signals, i.e. the detector will alternatelysignal the background alone or the total of signal plus background fromwhich the latter can be evaluated and compensated by sutracting thedifference. As an example the chopper can operate with a 30 slot at afrequency of 500 Hz.

The detector section 2 of the apparatus is shown applied against anorgan of the body to be investigated, for instance the ear lobe 13 in amanner such that the composite monochromatic beam 9 passes through thatorgan before reaching the detector section whereby it is attenuated bypartial absorption and partial diffusion in the tissues underexamination. As we have seen before, the main components of the bodytissues competing with glucose as light absorbers in the spectral regionof interest are the water and the proteins of the cells and interstitialfluid; however, the general distribution of these "background"constituents is fairly constant and so the general "shape" of thecorresponding spectrum superimposed to that of glucose is also ratherconstant including the bands with points the intensity of which issubstantially independent of the glucose concentration (isosbesticpoints). Therefore, as already mentioned, correlating the absorption ofthe background at the isosbestic points (wavelengths of reference) withthe effective thickness of the tissue layer of the organ underinvestigation traversed by the incident beam enables to determine thereference absorption factor used for normalizing the absorption datamade at the typical glucose λG wavelengths disclosed heretoforewherefrom the ultimate glucose concentration results are obtained.

In this connection, it should be noted that the principle of theaforementioned analysis can be expanded to analyze a three conponentmixture containing glucose, serum and water. Indeed, serum containsessentially all the dissolved constituents in blood or body fluid and,as mentioned above, several features in the absorption spectrum of serumare quite different from that of glucose. These features depicted fromthe curves of FIGS. 5 and 6 are emphasized in Table 1 below. Thereforethe concentration of glucose can be estimated from absorbancemeasurements using at least three different wavelength.

                  TABLE 1                                                         ______________________________________                                        Wavelength (nm)                                                                           Spectrum of serum                                                                           Spectrum of glucose                                 ______________________________________                                        1 574       flat          peak                                                1 732       peak          slope                                               1 765       dip           peak                                                2 052       peak          slope                                               2 098       dip           peak                                                2 168       peak          slope                                               2 269       slope         peak                                                2 291       peak          dip                                                 2 292       --            peak                                                ______________________________________                                    

By comparing FIG. 5 and FIG. 6, it is seen that very similar featuresstill exist in the two spectra. These features are the followings:

1 100 to 1 350 nm, flat portion and 2 223 nm, dip portion.

The detector section 2 comprises a light collecting integrating sphereor half-sphere (sometimes referred to as Ulbricht sphere) the wall ofwhich is layered with a dull high reflective coating for gathering andcollecting all available light penetrating through an opening 13a of thedevice which is directly applied against the organ under investigation(the ear-lobe in this embodiment). Materials which are highly reflectivein the 1 to 2.7 μm range are for instance Eastman 6080 white reflectancecoating containing barium sulfate or gold plating, the latter having abetter reflectance at the long wavelengths of the range. Using anintegrating full sphere is generally preferred unless a half-sphere isnecessary because of geometry considerations (see, for instance, themodification of FIG. 2). When this is required because of thepositioning of the device about the ear, the integrating sphere ishalved and its flat portion consists of a highly reflective mirror (goldcoating). The performance of the half-sphere of this construction issomewhat less than that of the full sphere but still acceptable becausethe mirror optically mimics a full sphere. Differently stated, a fullsphere is somewhat more efficient for collecting light but more bulky,so a compromise between sufficiently reduced physical size andsufficient efficiency is actually made in this embodiment. In thepresent drawing the curved portion of the half-sphere is presented ashaving ends somewhat flattened; however this should not be consideredphysically significant; the reason thereto being only of draftingconvenience. The light collected by multiple reflection in thehalf-sphere escapes through opening 13b and is condensed by means of acondensor 14 to fall on a detector 15. Any detector sensitive to therange of wavelengths used here can be used; an example of such detectoris a low temperature operating indium arsenide photodiode (JUDSONINFRARED INC. Pa 18936 USA) having the following properties.

    ______________________________________                                        Model                J 12-D                                                   Peak Wavelength      2.8 μm                                                Operating Temperature                                                                              77° K.                                            Time constant        0.5-2 μsec                                            Size                 2 mm (diameter)                                          Responsivity         1 A/W                                                    D                    4.10.sup.11 cmHz.sup.1/2 W.sup.-1                        Package              Metal Dewar                                              Liquid N.sub.2 Hold Time                                                                           6-8 hr                                                   Field of view        60°                                               ______________________________________                                    

Light collector means different from the integrating sphere can also beused in the present invention. Such means consist of an arrangement ofcurved surface mirrors internal reflective ellipsoid or paraboloidsurface portions) which collect the light exiting from the body tissuesand focus it onto the detecting means. Such light collecting means mayhave improved collecting efficiency over that of the integrating spherebecause of reduced number of reflections. Examples of collectingarrangements suitable for application in the invention can be found inthe following references: N. W. WALLACE, the Optical Layout of off-axisparaboles: Photonics Spectra, September 1984, p. 55; HARRICK SCIENTIFICCORP., Catalog HSC-83 (IR-Vis-UV accessories), Ossining, N.Y. 10562,USA.

The operation of the present apparatus is obvious from the previousdescription; for measuring the glucose in the ear tissue of a sittingpatient, the detector 2 is affixed onto the inside portion of the earlobe, for instance maintained by appropriate straps, and themonochromatic light from the source section 1 (usually mounted on anappropriate stand or rig on the side of the patient's chair) is directedon the external side of the ear portion directly facing the detector.The light beam 9 strikes the ear portion and after traversing itpenetrates into the collecting half-sphere 13 wherefrom it goes todetector 15 whereby it is converted into an electrical signal. The beam9 is interrupted regularly by the action of the chopper for the reasonsexplained before and, when in its non interrupted position, it providesan alternating dual or multiple wavelength incident light inputgenerated in the monochromator said incident light comprising at leastone measuring signal generally centered about the aforementioned valuesof 1575, 1765, 2100 or 2700 nm and at least one reference signal in thewide reference range or at the narrow wavelength ranges on both sides ofthe λG wavelengths. Thus the electrical signal obtained from detector 15is a multiplex signal repetitively carrying the information relative tothe optical apparatus background, the spectral background of the volumeof matter being analyzed and the glucose absorption measurementsaccording to a schedule under control of the chopper system 11 (line12b) and the computer circuits (line 12a). We shall see hereinafter howthis multiplex signal is decoded and processed.

Before doing so we shall turn to the modification of FIG. 2. Thismodification of which only a portion is represented in the drawingconstitutes an integrated light source-detector device to be placeddirectly over the skin, i.e. a device that operates according to theprinciple of reflection or back-scattering of light by tissues under theskin. The reference and measuring light signal generator of this deviceis similar to that used in the case of the device of FIG. 1 up to thechopper disk; therefore the light emerging from said chopper is giventhe same numeral 9 in FIG. 2.

The integrated light source-detector device of FIG. 2 is represented asbeing applied on the skin 20 of a patient; said skin being arbitrarilyrepresented by successive layers 20a, 20b and some underneath tissue20c. The present device comprises a movable mirror 21 which can bedisplaced horizontally continuously or stepwise while maintained inreflecting relationship with beam 9 so that the reflected beam 22 ispermanently directed into a horizontal slit 23 of the device. In orderto more clearly illustrate this point, a ghost image 21g of the mirror21 after being moved in a second position is provided on the drawing.The light beam 22 reflected by mirror 21 meets the skin at an incidenceangle indicated by λ. When the mirror is displaced in position 21g, itsorientation is such that the reflected beam 22g meets the skin at anangle β smaller than λ. The mechanical means to move and synchronouslytilt the mirror 21 are conventional and not represented here. Thepresent device further comprises as in the previous embodiment acollecting half-sphere 24 with an input opening 24a, a condensor 25 anda light detector 26 for converting the light gathered into an electricsignal represented by an arrow in the drawing.

The operation of the present device, which is fairly obvious from itsdescription, enables to undertake modulated depth glucose analysis belowthe skin. Indeed, during analysis, the mirror 21 can be moved back andforth so that the angle of penetration (α, β) of the beam 22 can bechanged at will. The angles of the corresponding penetrating beams 27and 27g will change accordingly and so will the position of theunderneath region under illumination wherefrom the back-scattered energywill be picked-up by the halfsphere entrance aperture 24a. This isclearly seen from the drawing in which the back-scattered light isindicated by numeral 28 when the excitation beam 22 falls at the angle αand by numeral 28g when the excitation beam 22g reaches the skin at anangle β. This technique permits the alternating exploration of differentzones at different depths under the skin whereby differentconcentrations of glucose can be determined or monitored for a period oftime. This is particularly useful for ascertaining the general shape ofthe background spectrum, i.e. the absorption of the medium in absence ofglucose or when the concentration of glucose is insignificant or of lowvariability as is the case in the superficial layer of the epidermis.i.e. the stratum-corneum. Thus, the measurement of the absorptionspectrum in the region 20a immediately under the skin surface willprovide reference results which may be continuously or periodicallycompared to corresponding results obtained from deeper layers of theepidermis or the dermis, whereby useful data about the concentration ofglucose in said deeper layers 20c can be obtained, this being directlyproportional to the blood glucose concentration. The construction of thepresent embodiment also enables to block the light directly reflected bythe skin surface at the impingement point. Indeed, such surfacereflected component is parasitic since it comprises no glucoseinformation and only contributes detrimentally to the background noiseas in the embodiments of the prior art. Another advantage of the presentinvention's embodiment is that it obviates or minimizes possibledisturbances caused by foreign substances contaminating the skin of theregion under examination.

The electric signals provided from detectors 15 or 26 are analyzed andprocessed in circuits which constitute also part of the apparatus of theinvention.

Such circuits shown on FIG. 3 comprise, starting from the photodetector15 or 26 (depending on whether the embodiments of FIG. 1 or 2 areconsidered), a preamplifier 30 (for instance a JUDSON INFRARED Model 700having an amplification of 10⁷ V/A) a gain programmable amplifier 31(for instance with gain varying from 1 to 200), an integrator 32 forholding and averaging over noise and an analog to digital converter 33(for instance a 16 bit unit). The integrator 32 is under control of atiming unit 34 timed by the clock of the chopper 11 (see FIG. 1).

The digital signal issuing from converter 33 comprising, in successionand according to a timing governed by said clock, the digitalizedinformation relative to the background noise, the glucose measurementsignal and the reference signals, is fed to a microprocessor 35 (forinstance an APPLE II microcomputer) also controlled by said clockwhereby the information is digested, computed according to a program ofchoice using one of the calculating methods disclosed heretofore anddisplayed or stored in terms of glucose determination data on either oneor more of a monitor 36, a printer 37 or a floppy disk recorder 38. Themicroprocessor 35 also provides the signal for timing and controllingthe wavelength scan or selection of the monochromator 8 (see line 12a).

REDUCTION TO PRACTICE

The following discloses a practical test effected according totransmissive technique (see FIG. 1). The data however apply equally wellto the reflective technique illustrated by the device of FIG. 2. Themeasurements were carried out against an aqueous reference backgroundsuch environment being sufficiently close to that overall body tissuesto be fully significant. General physical considerations over absorptionphenomena are also provided for reference.

The fundamental relation between optical absorbance and theconcentration of the absorbing material is given by the Beer-Lambertlaw.

    D=log.sub.10 (I.sub.o /I)=ε·C·L

where

D=optical density, absorbance.

I_(o) =intensity of incident light at wavelength λ.

I=intensity of light after passing through absorption cell.

C=concentration of the absorbing material (molar).

L=length of absorption path.

ε(λ)=extinction coefficient.

The validity of this relation is generally satisfactory if the radiationis monochromatic, if the concentrations of absorbing material are low,and if there are no significant molecular interactions, e.g.association, dissociation or structural changes for differentconcentrations. If the measurement phenomenon involves some significantdegree of scattering, the above relation is no longer strictly valid andcorrection factors must be introduced to restore its usefulness.Reference to such modification can be found in GUSTAV KORTUM'S bookReflexionsspektroskopie, SPRINGER Verlag (1969).

In the case of a mixture of m components, the Beer-Lambert law can begeneralized and expanded to include absorbance of each of the componentsat each analytical wavelength. ##EQU1## ε_(i) (λ)=specific absorbance ofa component i which is wavelength dependent

C_(i) =is the concentration defined as a mole fraction of the componenti, so that ##EQU2## I_(o), I and L are defined as before.

In the experiments reported below, an apparatus such as that describedwith reference to FIGS. 1 and 3 was used, the ear portion being replacedby glucose solutions in water (pure water was used as reference). Theparameters were: sample concentration of glucose=C2; concentration ofwater=C1 (C1+C2=1); path length for both pure water and solution=L;extinction coefficient of water=ε1; extinction coefficient ofglucose=ε2.

The absorbances can be written in the two cases as: ##EQU3##

The absorbance difference

    (ΔD=log (I.sub.H.sbsb.2.sub.O)-log (I.sub.glucose-solution)

can then be written as

    ΔD=D.sub.2 -D.sub.1

or

    ΔD=LC.sub.2 (ε.sub.2 -ε.sub.1)

and ##EQU4##

This equation shows that the concentration of glucose C₂ is proportionalto the absorbance difference ΔD in the two samples since the constantfactor L(ε₂ -ε₁) is known from operating conditions and kept constant.

As light of the incident intensity I_(o) passes alternately throughsamples 1 and 2 causing intensities I₁ and I₂, this can be written:

    C.sub.2 ˜D=log (I.sub.o /I.sub.1)-log (I.sub.o /I.sub.2)=log (I.sub.2 /I.sub.1)=log I.sub.2 -log I.sub.1                        (1)

This means that it is sufficient to measure the difference of theabsorbance in the samples 1 and 2. The incident intensity I_(o) need notbe measured.

Thus, the following three detected signals are processed in themicroprocessor 35. (The proportionality constant between light intensityand detector signals is g).

S_(B) =gB: background when there is no light falling onto the samples. Bis the background equivalent light intensity from ambient light plus thedetector noise.

S₁ =g(I₁ +B): signal caused by test sample 1=(intensity I₁ plusbackground).

S₂ =g(I₂ +B): signal caused by reference sample 2=(intensity I₂ plusbackground).

For each sample, the difference between signal and background is taken,resulting in ΔS₁ and ΔS₂.

sample 1: ΔS₁ =g(I₁ +B)-gB=gI₁

sample 2: ΔS₂ =g(I₂ +B)-gB=gI₂

These operations were synchronized by the chopper system (500 Hz) toeliminate drifts of the background (zero) signal B which normally occurat very slow rate.

The quantities ΔS₁ and ΔS₂ were measured automatically for a number oftimes (100).

To find out the glucose concentration in sample 2, the absorbance valuefrom water in sample 1 was used as a reference and the two values weresubtracted from each other (see equation (1)):

    ΔD=log I.sub.2 -log I.sub.1 =log (ΔS.sub.2 /g)-log (ΔS.sub.1 /g)

    ΔD=log ΔS.sub.2 -log ΔS.sub.1            (2)

The equation shows that the actual light intensities in equation 1 canbe replaced by the electrical detector signals. The result is notdependent on the proportionality constant g.

The data processing program used in this embodiment is also able tocompute the errors of a set of measurements by using classical algebraicequation of error propagation theory.

Four different glucose concentrations 0M, 0.05M, 0.5M and 1M and twodifferent wavelengths 1100 nm (reference wavelength λR) and 2098 nm(test wavelength λG) were chosen.

At 1100 nm, the glucose spectrum is flat. The water absorbance has itslowest value. At about 2100 nm (more precisely 2098 nm), the glucosespectrum exhibits a characteristic absorption peak. The water absorbancehere is about two absorbance units higher than an 1100 nm.

In FIG. 4, the absorbance values ΔD are plotted for the two differentwavelengths as a function of glucose concentration. The absorbance curvefor 1100 nm falls slightly with concentration of glucose. As glucose hasno specific absorption at this wavelength, essentially water ismeasured. With increasing glucose concentration, the water concentrationis correspondingly reduced, so that the absorbance measured becomessmaller. At 2098 nm, a strong increase in absorbance with glucoseconcentration is observed. The curve is the result of two oppositeacting effects: the reduced concentration of water causes an absorbancereduction which is similar to that for 1100 nm; the strong glucoseabsorbance at this wavelength causes an absorbance increase. As theglucose absorbance is about 13 times stronger, the result is an increasein absorbance. Thus, the net effect of glucose absorbance isapproximately the vertical difference A between the curve for 2098 nmand the curve for 1100 nm. This confirms the soundness of the twowavelength methods embodiment, one at 2098 nm and the other at thereference independent wavelength of 1100 nm. Of course, similar methosusing the other wavelengths disclosed in this specification are alsopossible and provide comparable results

In addition to the aforementioned computations of measured results toestimate the concentrations of glucose, serum and water from absorbancemeasurements at various wavelengths, two other procedures for improvedaccuracy can be considered, i.e. regression and clustering. Detailsincluding differences and advantages of each technique are explained inthe following reference and the references cited therein: J. B. Gayle,H. D. Bennett, "Consequences of Model Departures on the Resolution ofMulticomponent Spectra by Multiple Regression and Linear Programming",Analytical Chemistry 50, 14, December 1978, p. 2085-2089. Clusteringallows processing of more than one parameter (e.g., absorbance and skindepth). The starting clusters need not be data from pure solutions butcan be mixtures. This is an advantage because serum normally alwayscontains glucose so that reference data from serum without glucose aregenerally not easily obtainable.

Thus, in the case of a multi-component mixture, the main components ofthe body tissues competing with glucose as light absorbers in thespectral region of interest have characteristic absorptions distinctfrom the aforementioned selected typical glucose absorptions. Further,they also differ from glucose by their absolute concentration and by thetime constant of their concentration variation. However, the generaldistribution of those "background" constituents is fairly constant.Therefore the ultimate glucose concentration can be obtained fromabsorbance measurements at various wavelengths.

We claim:
 1. A spectrophotometric apparatus for determining the glucoseconcentration in body tissues transcutaneously and non-invasively,comprising:(a) a directional optical light source located external tothe body, the spectral composition of the beam of light from said sourcebeing such that it can penetrate the skin to tissues below; (b) meansfor collecting light transmitted or diffusely reflected from saidirradiated tissue; (c) means for detecting and converting intoelectrical signals light gathered from at least one band with ameasuring signal wavelength λG of 1575, 1765, 2100 or 2270 + or - 15 nm,typical of the glucose absorption spectrum, and at least one band with areference signal wavelength λR in the range of 1000 to 2700 nm, typicalof the absorption spectrum of background tissue containing glucose butin which the absorption of glucose is nil or insignificant; and (d)means for transforming said electrical signals into data representingglucose determinations.
 2. The apparatus of claim 1, further comprisinga means for varying continuously or stepwise the incidence angle of saidbeam of light relative to the body surface, so that the depth under theskin surface wherefrom the light is gathered after absorption is varied.3. The apparatus of claim 2, wherein said means for varying saidincident angle comprises a mirror displacable in reflective relationshipwith said beam and positioned so that the reflected beam is alwaysdirected toward the same point on the skin.
 4. The apparatus of claim 1,wherein said collecting means comprises the internal reflective surfaceof a halfsphere, said surface being coated with gold and/or a layer ofbarium sulfate containing paint.
 5. A spectrophotometric method for thetranscutaneous, non-invasive determination of glucose concentrations inbody tissues, comprising the steps of:(a) irradiating a selected bodyportion with light from a directional optical lamp source; (b)collecting the resulting luminous energy (I) either transmitted ordiffusively reflected by a sample volume of body tissue under the skinof said irradiated body portion, said collected light including at leastone band with a measuring signal wavelength λG of 1575, 1765, 2100 or2270 + or - 15 nm, typical of the glucose absorption spectrum, and atleast one band with a reference signal wavelength λR in the range of1000 to 2700 nm, typical of the absorption spectrum of background tissuecontaining glucose but in which the absorption of glucose is nil orinsignificant; (c) converting said collected light into electricalsignals IG and IR representing said measuring and reference bands,respectively; and (d) entering said electrical signals into anelectronic computer for transformation into glucose concentrations. 6.The method according to claim 5, wherein a normalizing factor isestablished from the difference in absorption in said reference bandwhen glucose is present and when glucose is absent or in insignificantquantities, absorbance values for the glucose as measured in saidmeasuring band are normalized with said factor and said normalizedvalues are used for said glucose determination.
 7. The method accordingto claim 6, wherein said reference band corresponds to an isosbesticpoint selected in the range of 1100 to 1300 nm or in the regionsstraddling said λG bands.
 8. The method according to claim 6, whereinthe normalizing factor is established by alternately effectingabsorption in said reference band first in a portion of body tissuewhere the amount of glucose is low or insignificant and, second, in aregion of tissue in which the glucose concentration is to be analyzed.9. The method according to claim 5, wherein said IG and IR signals aredifferentiated with respect to λ within the area of said measuring andreference bands, respectively, the difference between the differentialsbeing representative of said glucose determination.